Inorganic phosphate (Pi) is involved in a large number of biological processes and it is desirable to be able to measure the concentration of Pi and the changes in such concentration in biological systems. Phosphate assays, which measure Pi concentration, are useful in a number of diagnostic methods, as well as in research into the functioning of biological systems.
Enzymatic phosphate assays are based on a phosphate-requiring enzyme, often a phosphorylase. Reference 1 describes a method in which a purine-nucleoside phosphorylase is used to convert a nucleoside (inosine) to ribose-1-phosphate and a base, in this case, hypoxanthine. The hypoxanthine is then converted into a coloured agent, from which the extent of inosine conversion, which is dependent upon Pi concentration, may be determined.
Enzymatic phosphate assays tend to be relatively insensitive. For example, reference 2 describes a method that may not be used below Pi concentrations of 2 μM. Furthermore, although more rapid than chemical phosphate assays, enzymatic phosphate assays are generally too slow to allow the study of kinetics of many biological systems in real time.
A number of phosphate assay systems are known in the art. For example, Malachite Green Phosphate Detection (MGPD) kits are useful for the quantitative detection of Pi. The Quantichrom Phosphate Assay Kit (BioAssay Systems) is one such MGPD kit. However, the assay used is very slow requiring incubation to achieve colour development. Furthermore, MGPD kits are generally useful only at high concentrations of phosphate (approximately 0.3 mM-50 mM).
The EnzChek Phosphate Assay Kit from Invitrogen (Molecular Probes) has a phosphate concentration detection range of 2 μM-150 μM and a workable pH range of 6.5-8.5 (taken from data sheet). Again, this test is unsuitable for the detection of low phosphate concentration.
A number of proteins are known which specifically bind to Pi. For example, transport of Pi into and out of cells and organelles is executed by specific transport proteins. In bacterial cells, this is achieved by way of a high affinity transport system dependent on a phosphate-binding protein. Such proteins are able to specifically recognise inorganic phosphate, bind to it and transport it across cell membranes or between cellular compartments.
An example of such a protein is the E. coli phosphate binding protein (PBP) which is encoded by the phoS gene of E. coli. This protein is located in the periplasm of E. coli as part of the Pi scavenging system of the bacterium, which operates under conditions of Pi starvation, and its binding affinity for Pi is very high. The phoS gene has been cloned and sequenced [3,4]. Moreover, it has been determined that PBP binds Pi tightly, and the crystal structure of the Pi-bound form has been solved to high resolution [5], as has the structure of a Pi-free form [6]. These studies have shown PBP to be a monomeric protein of 35 kD separated into two domains, with a Pi-binding cleft between them. The Pi-binding cleft moves between open and closed positions on Pi binding.
Reference 7 describes the modification of PBP to introduce a coumarin label at the edge of the Pi-binding cleft. The conformational change to the binding cleft which occurs upon phosphate binding is translated into an increase in the fluorescence of the coumarin label. However, the universality of phosphate in biological systems and the desire to monitor the kinetics of biological and chemical processes which involve the consumption or production of Pi makes the development of further and improved phosphate assays important.